1. Field of Invention
This invention relates to novel reagent compositions and to methods for their preparation. In particular, it relates to novel lyophilized reagent spheres and diluents useful in the analysis of biological samples.
In preparing reagents for convenient and efficient testing of clinical biological samples, it is frequently important to obtain dry chemical blends in uniform, discrete amounts. These reagents must be efficiently and economically prepared in small precisely measured quantities. Reagents comprising organic materials, however, tend to spoil or degrade on storage, thus creating quality control problems. Thus, reagents are typically provided in dried form to increase stability. Current technology for producing dry chemical blends involves procedures such as dry blending, spray drying, or fluid bed drying. All three of these procedures, however, have limitations that make them costly, inefficient or difficult to carry out.
In dry blending technology, it is difficult to obtain homogeneous blends of chemicals that have different densities. Moreover, homogeneity is particularly difficult to achieve when very small amounts of ingredients are mixed with large amounts of others. Once made homogeneous, it is extremely difficult to reproducibly (within 1 percent) dispense small amounts (less than about 10 mg) of the blended chemicals.
Spray drying technology provides more homogenous blends of chemicals because the reagents are first dissolved in liquid. Using spray drying, however, it is difficult and costly to obtain precisely sized amounts of blended chemicals. As generally practiced, this process yields particles with size distributions having coefficients of variation greater than 20 percent. The resulting particles have to be reprocessed (usually agglomerated) to obtain uniform particle sizes. After agglomeration, the particles are generally less soluble than the original spray dried particles. Moreover, these procedures typically use fluorocarbon cryogenic solutions which are hazardous to the environment.
Fluid bed technology relies upon spraying a liquid reagent blend onto a particle and drying the liquid to obtain a particle coated with the blended reagents. Using this procedure, it is difficult to obtain uniformly sized particles and to produce a uniform coating.
Of particular interest to the present invention are reagents useful in analyzing biological samples, such as blood plasma or serum, in centrifugal analyzers. The rotors used in such analyzers measure volumes of the sample to be tested, mix the sample with an appropriate diluent and separate fluid from cellular components. The rotors also provide a plurality of separate test wells containing chemical reagents in which discrete volumes are optically tested.
Analysis of biological samples in the test wells of centrifugal rotors impose a number of requirements on the reagents used for analysis. In particular, because the analysis is typically highly automated, speed of analysis is at a premium. In addition, many clinical diagnostic analyses require that measurements be made within a short time after the sample is added to the reagent. Thus, the dried reagent preparations must dissolve quickly in the sample solution. In addition, rapid rehydration of the reagents can cause bubble formation, which adversely affects results by interfering with optical measurement.
In centrifugal analyzers, the sample is typically mixed with a diluent before analysis. It is not possible to directly measure the amount of diluent added while the diluted sample is in the rotor. Obviously, improperly diluted samples will produce erroneous results. Thus, convenient methods for determining amount of dilution of the sample in situ are required. In addition, if the sample to be diluted comprises cells, the diluent must contain isotonic concentrations of compounds to prevent osmotic shock to the cells. Such compounds, however, must not enhance or inhibit any of the analyses. Many isotonic solutions are disclosed in the prior art, including saline, glucose, or phosphate buffered saline solutions. None of these solutions are suitable because they can affect results, because they provide additional buffering capacity to the solution or because they add chemicals which are the same as the analytes of interest.
The prior art thus lacks reagent compositions which avoid the above problems in centrifugal analyzers. In particular, the prior art lacks economical and reliable reagent preparations which dissolve quickly in sample solutions and avoid bubble formation. Moreover, currently available diluents are not suitable because dilution cannot be easily measured and they can alter the results of the analysis. The present application addresses these and related problems.
2. Description of Background Art
U.S. Pat. Nos. 3,721,725 and 3,932,943 relate to methods for producing lyophilized reagents comprising spraying a solution containing the reagents into a moving bath of fluorocarbon refrigerants and lyophilizing the resultant frozen droplets. 4,848,094 discloses methods for the generation of essentially spherical frozen droplets and improved methods for removing frozen droplets from a cryogenic liquid. 4,655,047 describes methods for freezing drops of relatively thick liquids by dropping them from a small height into a cryogenic material. 3,819,488 provides stable lyophilized diagnostic compositions for determining glutamic oxalic transaminase and glutamic pyruvic transaminase activities. 4,588,696 relates to preparation of tablets used in testing for formaldehyde and/or glutaraldehyde. 4,295,280, 4,351,158, and 4,712,310 all relate to methods for preparing homogenous preparations comprising compounds which are incompatible. 4,820,627 discloses a fluidized bed process for preparing particles suitable for tableting into diagnostic reagents. 4,115,537 relates to diagnostic tablets containing ion exchange resins. 4,755,461 is directed to tableted blood plasma compositions. 4,678,812 and 4,762,857 both relate to diagnostic tablets comprising trehalose as an excipient and stabilizer. The use of TRITON.RTM.X-100 is also disclosed. 4,716,119 discloses the addition of tetramethylammonium acetate to blood serum. Romanian Patent Appln. No. 85,155 relates to enzymatic alkaline phosphotase reagent tablets comprising p-nitrophenyl phosphate. Driscoll et al., Clin. Chem., 29:1609-1615 (1983) discloses an instrument/reagent system comprising tableted reagents for performing photometric assays.